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WO2009073643A2 - Revêtements à libération d'oxyde nitrique incorporant une enzyme oxyde nitrique synthase - Google Patents

Revêtements à libération d'oxyde nitrique incorporant une enzyme oxyde nitrique synthase Download PDF

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Publication number
WO2009073643A2
WO2009073643A2 PCT/US2008/085224 US2008085224W WO2009073643A2 WO 2009073643 A2 WO2009073643 A2 WO 2009073643A2 US 2008085224 W US2008085224 W US 2008085224W WO 2009073643 A2 WO2009073643 A2 WO 2009073643A2
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Prior art keywords
nos
coating
polymeric material
layer
nitric oxide
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WO2009073643A3 (fr
Inventor
Mekki Bayachou
Charbel Abou Diwan
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Cleveland State University
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Cleveland State University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
    • A61L2300/114—Nitric oxide, i.e. NO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents

Definitions

  • the presently disclosed embodiments are directed to the field of biocompatible coatings comprising Nitric Oxide Synthase enzyme, and related biocompatible matrices comprising this enzyme.
  • Nitric oxide a molecule generated by the enzyme Nitric Oxide Synthase (NOS) from arginine, is known as a potent anti-platelet agent.
  • NO generated from endothelial cells inhibits platelet adhesion, aggregation, and further recruitment of platelets to the growing thrombus.
  • Prior artisans have incorporated various nitric oxide releasing compounds in coatings that are deposited on or about various medical devices in an attempt to reduce the potential for thrombus formation. Although varying degrees of success have been achieved, as far as is known, this strategy suffers from the fact that the release of NO is limited. As a result of depleting the NO source from the coating, resulting concentrations of NO are also typically deficient and particularly so after an extended time period.
  • Identification of such NO releasing materials will provide enhanced biocompatibility for a wide range of materials typically used in preparing implantable devices and will likely lead to applications of new materials. Furthermore, deleterious or undesirable side effects, associated with the implantation process, could be avoided, and better clinical outcomes achieved. It is also expected that such NO releasing materials will be equally applicable in the fields of implantable prosthetic grafts and implantable sensors.
  • the difficulties and drawbacks associated with previous practices and methods are overcome in the present methods, items and devices that release nitric oxide, or that induce release of nitric oxide, in humans.
  • the present invention can be in a variety of different forms and used in many different ways. However, the invention is preferably provided and used as a coating on implantable and/or extracorporeal medical devices.
  • the present invention provides a method for producing a nitric oxide release coating on a substrate.
  • the substrate may be associated with any item or device.
  • An example of such a substrate is an outer surface of an implantable medical device.
  • the method comprises providing a substrate defining an exposed surface.
  • the method also comprises depositing a polymeric material on the exposed surface of the substrate.
  • the method includes forming a NOS-coating precursor on the deposited polymeric material. And, the method then includes drying the resulting array to produce the nitric oxide release coating.
  • the present invention provides a method for releasing nitric oxide within a human body.
  • the method comprises forming a nitric oxide release coating on a substrate, wherein the coating comprises at least one layer including NOS and at least one layer including a polymer.
  • the method also comprises introducing the coated substrate into the human body.
  • the present invention provides an item adapted for use within a human body.
  • the item defines an exterior surface and comprises a multi-layer coating disposed on the exterior surface.
  • the multi-layer coating includes at least one layer of a polymeric material and at least one NOS-containing layer.
  • the present invention provides a method for treating a human having a biological disorder treatable with nitric oxide.
  • the method comprises administering to the human, a medical device comprising a multilayer coating disposed on at least a portion of the device.
  • the multi-layer coating includes at least one layer of a polymeric material and at least one NOS-containing layer.
  • the present invention provides a method for treating or more particularly deterring, microorganism growth on an implantable medical device.
  • the method comprises depositing a polymeric material on an exposed surface of the device.
  • the method also comprises forming a NOS-coating precursor on the deposited polymeric material.
  • the method additionally comprises drying the resulting layered array to produce a nitric oxide release coating that serves to reduce the extent of microorganism growth on the medical device.
  • Figure 1 is a graph of wavelength of UV and visible light and corresponding absorption spectra of iNOS at the surface of modified ITO slides according to a preferred embodiment of the present invention.
  • Figure 2 is a graph illustrating the IR spectrum of iNOS enzyme within a preferred embodiment PEI film.
  • Figure 3 is a graph illustrating catalytic reductions of NO by PEI/NOS in pH 7.0 for different NO concentrations.
  • Figure 4 is a graph illustrating resonant frequency F 0 change during PEI/NOS LBL deposition at the surface of a quartz crystal.
  • Figure 5 is a three dimensional AFM image of the deposition of a layer of polymer PEI at the surface of a HOPG slide.
  • Figure 6 is a three dimensional AFM image of the deposition layer of NOS over a layer of the polymer PEI at the surface of the HOPG slide of Figure 5.
  • Figure 7 is a three dimensional AFM image of the same HOPG slide after the sequential deposition of PEI over a layer of NOS shown in Figure 6, resulting in a preferred embodiment PEI/NOS/PEI coating.
  • Figure 8 is a graph illustrating total surface NO flux from additional preferred embodiment PEI/NOS five layer assemblies.
  • Figure 9 is a graph illustrating NO surface flux time as compared to time from preferred embodiment PEI/NOS five layer assemblies.
  • Figure 10 is a graph illustrating measured NO fluxes after 8 hours with various thicknesses of preferred embodiment coatings.
  • Figure 11 is a phase contrast microscopy image of pre-incubation of a PEI/BSA film.
  • Figure 12 is a phase contrast microscopy image of pre-incubation of a preferred embodiment PEI/NOS film.
  • Figure 13 is a phase contrast microscopy image of pre-incubation with platelet rich plasma of the PEI/BSA film.
  • Figure 14 is a phase contrast microscopy image of pre-incubation with platelet rich plasma of the PEI/NOS film.
  • Figure 15 is a graph of an LDH assay of platelet adhesion on the surface of various preferred embodiment PEI/NOS coatings.
  • Figure 16 is a graph of the percentage platelet adhesion on the surface of the PEI/NOS coatings referenced in Figure 15.
  • the present invention relates to coatings and their use that can for example, be applied to various implantable medical devices.
  • the invention also relates to medical devices comprising such coatings.
  • the present invention relates to various uses and application of the coatings.
  • the coatings are biocompatible and include an enzyme, Nitric Oxide Synthase (NOS), which provides a source of nitric oxide (NO) that functions to decrease platelet adhesion, enhance fibroblast proliferation, and decrease microbial growth.
  • NOS Nitric Oxide Synthase
  • the coatings are formed or otherwise deposited along the exterior surface of an implanted or implantable device.
  • the enzyme NOS can be incorporated into the biocompatible coatings by a variety of techniques, however, a preferred process is via layer-to-layer electrostatic adsorption.
  • a significant feature of the present invention is that incorporation of the NOS enzyme into the coatings mimics the nitric oxide generating behavior of endothelial cell linings.
  • the present invention enables comparable in vivo concentration levels for nitric oxide to be achieved.
  • the present invention enables the continuous production of nitric oxide from the coatings and in effective concentration levels, as opposed to prior art attempts in which compounds or nitric oxide precursors were incorporated into device coatings, which inevitably resulted in depletion of nitric oxide from the coating or ineffective concentration levels.
  • the enzyme Nitric Oxide Synthase is incorporated into a biocompatible polymeric matrix by a process involving layer-by- layer (LBL) electrostatic adsorption to construct a multi-component protein film that mimics the NO-generating behavior of the endothelial cell lining.
  • LBL layer-by- layer
  • the layer-by-layer electrostatic method and the polymer matrix used to build the coating are described herein to illustrate the benefits of the present invention and provide examples of actual performance of the invention. However, it is to be understood that the present invention includes the use of other methods and materials. Accordingly, the present invention is not limited to the methods and materials described herein.
  • a NOS-based coating in polymeric and other biocompatible matrices provides a source of NO, which utilizes endogenous compounds to maintain a continuous supply of NO at levels that are in the range of endothelial cells. Furthermore, as described herein, by altering the coating composition, control over the NO release levels and rates of release can be readily achieved. In addition, as described herein, the present invention coatings decrease platelet adhesion in vitro. Moreover, the present invention coatings serve to decrease or reduce the potential for microbial growth on or in the vicinity of the coating(s).
  • Nitric oxide has a wide variety of biological functions including being a potent antiplatelet agent, inhibiting smooth muscle cell proliferation, preventing microbial growth, and enhancing wound healing.
  • NO nitric oxide
  • Numerous approaches are currently being investigated in an attempt to develop polymeric materials that are more blood-compatible. In general, these approaches can be categorized into two main trends: first, methods that mimic endothelial cells' anti-thrombogenic properties, and, second, methods that use modified chemical surfaces and added moieties that exhibit decreased protein and cell adhesion.
  • Nitric oxide, thrombomodulin, prostacyclin, and heparans contribute to the non-thrombogenic properties of the endothelial cells.
  • NO donors e.g., diazeniumdiolates or S- nitrosothiols (RSNOs)
  • RSNOs S- nitrosothiols
  • NO-releasing materials have also the potential to inhibit restenosis following angioplasty. NO exhibits a short half-life, typically less than 1 second, in the presence of oxygen and hemoglobin. No systemic effects should be caused by NO-releasing coatings because all NO released will act and be rapidly consumed locally near the surface of the device.
  • N- diazeniumdiolate based NO-releasing polymers Two classes have previously been explored.
  • N-diazeniumdiolates are inorganic NO donors formed by the reaction of a secondary amine structure with two moles of NO gas under high pressure, creating a relatively stable adduct structure.
  • a counteraction is required to fulfill electroneutrality of the negatively charged diazeniumdiolate adduct, leading to zwitterionic molecules.
  • diazeniumdiolates based NO-releasing polymers Three general structural types of diazeniumdiolates have been outlined for preparing diazeniumdiolates based NO-releasing polymers: Dispersed non-covalently bound small molecules where the diazeniumdiolate group is attached to amines in low molecular weight compounds; covalently bound diazeniumdiolates group to polymeric side chains; or those in which the diazeniumdiolates group is bound to the polymeric backbone.
  • the second class of NO donors are S-nitrosothiols. They are believed to serve as a NO reservoir and transporter within biological systems. S-Nitroso-albumin and S-nitrosoglutathione are the most abundant naturally occurring S-nitrosothiols circulating in blood. The cleavage of the S-NO bond releases NO by three known mechanisms: copper mediated decomposition, the direct reaction of ascorbate, and photolytic decomposition.
  • NOS is an enzyme in the body that contributes to transmission from one neuron to another, to the immune system, and to dilating blood vessels. It does so by synthesis of nitric oxide (NO) from the terminal nitrogen atom of L-arginine in the presence of NADPH and dioxygen (O 2 ).
  • NOSs is a family of related enzymes encoded by separate genes. NOS is one of the most regulated enzymes in biology. There are three known isoforms, two are constitutive (cNOS) and the third is inducible (iNOS). Cloning of NOS enzymes indicates that, cNOS include both brain constitutive (NOS1 or nNOS) and endothelial constitutive (NOS3 or eNOS), and the third is the inducible (NOS2 or iNOS) gene. The different forms of NO synthase have been classified as follows:
  • Neuronal NOS produces NO in nervous tissue in both the central and peripheral nervous system. Neuronal NOS also performs a role in cell communication and is associated with plasma membranes. nNOS can be inhibited by NPA (N-propyl-L-arginine). This form of the enzyme is specifically inhibited by 7- nitroindazole.
  • NPA N-propyl-L-arginine
  • Inducible NOS can be found in the immune system but is also found in the cardiovascular system. It uses the oxidative stress of NO (a free radical) to be used by macrophages in immune defense against pathogens.
  • Endothelial NOS also known as nitric oxide synthase 3 (NOS3), generates NO in blood vessels and is involved with regulating vascular function.
  • NOS3 nitric oxide synthase 3
  • a constitutive Ca 2+ dependent NOS provides a basal release of NO.
  • eNOS is associated with plasma membranes surrounding cells and the membranes of Golgi bodies within cells.
  • All three isoforms (each of which is presumed to function as a homodiner during activation) share a carboxyl-terminal reductase domain homologous to the cytochrome P450 reductase. They also share an amino-terminal oxygenase domain containing a heme prosthetic group, which is linked in the middle of the protein to a calmodulin-binding domain. Binding of calmodulin appears to act as a "molecular switch" to enable electron flow from flavin prosthetic groups in the reductase domain to heme. This facilitates the conversion of O 2 and L-arginine to NO and L-citrulline.
  • the oxygenase domain of each NOS isoform also contains a BH 4 prosthetic group, which is required for the efficient generation of NO.
  • BH 4 activates heme-bound O 2 by donating a single electron, which is then recaptured to enable nitric oxide release.
  • the endothelial isoform of the enzyme Nitric Oxide Synthase, eNOS is a key component of the endothelial cell lining. It was reported that eNOS-over- expressing endothelial cells seeding of synthetic small diameter vascular grafts decreased human platelet aggregation by 46% and bovine aortic smooth muscle cell proliferation by 67.2% in vitro. In vivo studies involving production of high levels of NO from NOS showed the ability to prevent platelet adhesion.
  • the present invention provides a polymeric coating that comprises NOS, and which is preferably applied to the exterior of implantable medical devices.
  • the term "medical device” as used herein refers to any device having surfaces that contact tissue, blood, or other bodily fluids in the course of their use or operation, which are found on or are subsequently used within a mammal and typically, humans. Medical devices include, for example, extracorporeal devices for use in surgery, such as blood oxygenators, blood pumps, blood storage bags, blood collection tubes, blood filters including filtration media, dialysis membranes, tubing used to carry blood and the like which contact blood which is then returned to the patient or mammal.
  • Medical devices also include endoprostheses implanted in a mammal (e.g., a human), such as vascular grafts, stents, pacemaker leads, surgical prosthetic conduits, heart valves, and the like, that are implanted in blood vessels or the heart.
  • Medical devices also include devices for temporary intravascular use such as catheters, guide wires, amniocentesis and biopsy needles, cannulae, drainage tubes, shunts, sensors, transducers, probes and the like which are placed into the blood vessels, the heart, organs or tissues for purposes of monitoring or repair or treatment.
  • Medical devices also include prostheses such as artificial joints such as hips or knees as well as artificial hearts.
  • medical devices include penile implants, condoms, tampons, sanitary napkins, ocular lenses, sling materials, sutures, hemostats used in surgery, antimicrobial materials, surgical mesh, transdermal patches, and wound dressings/bandages for example.
  • Medical sutures are another example of a "medical device" as that term is used herein and typically are made from synthetic polymers such as nylon, polytetrafluoroethylene, polyester, polyethylene, polypropylene, polyglycolic acid, or polyglactin 910 which can be monofilament or many filaments twisted together, spun together, or braided.
  • a preferred embodiment of the present invention is a suture filament that can be coated with an NO-releasing polymeric composition of the present invention such that the wound is closed, yet the local release of NO can prevent or reduce platelet aggregation, thereby preventing or minimizing a blockage.
  • an NO-releasing polymeric composition can be used in conjunction with any suitable suture material to prepare an NO-releasing suture filament.
  • suture thread e.g., nylon
  • a nitric oxide-releasing composition of the present invention could be twisted, spun, or braided together with a nitric oxide-releasing composition of the present invention to prepare a multifilament suture. The primary requirements are that the resulting suture thread maintains the desired level of NO release, strength, elasticity, and non-reactivity with bodily tissue.
  • the substrates typically utilized for implantable medical devices receive the coatings described herein.
  • the outer surface or exterior of a medical device constitutes the substrate as described herein.
  • the substrate can be of any suitable biocompatible material, such as metal, glass, ceramic, plastic, or rubber, or combinations thereof.
  • the substrate is metal.
  • the substrate used in the preparation of the medical device can be derived from any suitable form of a biocompatible material, such as, for example, a sheet, a fiber, a tube, a fabric, an amorphous solid, an aggregate, dust, or the like.
  • Metal substrates suitable for use in the present invention include, for example, stainless steel, nickel, titanium, tantalum, aluminum, copper, gold, silver, platinum, zinc, Nitinol, inconel, iridium, tungsten, silicon, magnesium, tin, alloys, coatings containing any of the above, and combinations of any of the above. Also included are such metal substrates as galvanized steel, hot dipped galvanized steel, electrogalvanized steel, annealed hot dipped galvanized steel, and the like.
  • the metal substrate is stainless steel.
  • Glass substrates suitable for use in the invention include, for example, soda lime glass, strontium glass, borosilicate glass, barium glass, glass-ceramics containing lanthanum as well as combinations thereof.
  • Ceramic substrates suitable for use in the invention include, for example, boron nitrides, silicon nitrides, aluminas, silicas, combinations thereof, and the like.
  • Plastic or polymeric substrates suitable for use in the invention include, for example, acrylics, acrylonitrile-butadiene-styrene, acetals, polyphenylene oxides, polyimides, polystyrene, polypropylene, polyethylene, polytetrafluoroethylene, polyvinylidene, polyethylenimine, polyesters, polyethers, polyamide, polyorthoester, polyanhydride, polyether sulfone, polycaprolactone, polyhydroxy-butyrate valerate, polylactones, polyurethanes, polycarbonates, polyethylene terephthalate, as well as copolymers and combinations thereof.
  • Typical rubber substrates suitable for use in the invention include, for example silicones, fluorosilicones, nitrile rubbers, silicone rubbers, fluorosilicone rubbers, polyisoprenes, sulfur-cured rubbers, butadiene-acrylonitrile rubbers, isoprene-acrylonitrile rubbers, and the like.
  • the substrate could also be a protein, an extracellular matrix component, collagen, fibrin or another biologic agent or a mixture thereof. Silicones, fluorosilicones, polyurethanes, polycarbonates, polylactones, and mixtures or copolymers thereof are preferred plastic or rubber substrates because of their proven bio- and hemocompatability when in direct contact with tissue, blood, blood components, or bodily fluids.
  • Other suitable substrates include those described in WO 00/63462 and U.S. Patent No. 6,096,070.
  • stents are a prime example of a “medical device” that can receive the coatings described herein.
  • the metals used to manufacture stents are typically 316L stainless steel (316L SS), platinum-iridium (Pt-Ir) alloy, tantalum (Ta), nitinol (Ni-Ti), cobalt-chromium (Co-Cr) alloy, titanium (Ti), pure iron (Fe), and Magnesium (Mg) alloys.
  • Stents are also typically coated with coatings such as inorganic coatings, coatings of endothelial cells, and porous materials.
  • inorganic coatings include, but are not limited to gold, iridium oxide, silicon carbide (SiC), and diamond-like carbon.
  • SiC silicon carbide
  • a non-limiting example of a porous coating material used on stents is segmented polyurethane film. These coatings can be used in conjunction with the present invention coatings, or may serve as the substrate upon which is applied, the coatings described herein.
  • any type of polymer can be used in the present invention coatings so long as it exhibits suitable properties that enable it to be used in association with a biological system and preferably implanted in a human and hence "biocompatible,” and so long as it is compatible with NOS.
  • All of the formulations of the present invention utilize one or more biocompatible polymeric compounds.
  • polymer and “polymeric” are, unless otherwise indicated, intended to broadly include homopolymers and block/random copolymers (and oligomers) including a chain of at least three or more monomer structural units formed by polymerization reactions (e.g., condensation or ring-opening polymerization).
  • Preferred biocompatible polymers are preferably formed by a condensation type polymerization.
  • the biocompatible polymers are homopolymers, while for others they are copolymers.
  • the repeating structural units comprise amide units, ester units, or mixtures thereof.
  • Preferred such biocompatible polymers include at least one chain of units of the formula — [X— R 1 — C(O)] — wherein each R 1 is an independently selected organic group that links the X group to the carbonyl group; and each X is independently oxygen, sulfur, or catenary nitrogen.
  • Such compounds can include chains having different R 1 groups, although for certain embodiments each R 1 moiety is the same.
  • the preferred X group is oxygen.
  • Particularly preferred biocompatible polymers are relatively low molecular weight polylactic acids (PLAs).
  • lactic acid is well known to be endogenous in humans, highly biocompatible and, therefore, desirable from a regulatory approval standpoint.
  • Other biocompatible polymers are also useful in methods and formulations according to the present invention.
  • homopolymers and copolymers of lactic acid, glycolic acid, trimethylene carbonate, hydroxybutyric acid, and p-dioxanone have all been found to be particularly useful in various embodiments of the present invention.
  • polydioxanone and polylactic-co-glycolic acids are well established as being biocompatible and, accordingly, are also good candidates from a regulatory approval standpoint.
  • suitable polymers include biostable polymers such as polyethylene terephtalate (PET), polyurethane (PU), and silicone; biodegradable polymers such as poly-L-lactic acid (PLLA), polyorthoester, polycaprolactone, and polyethylene oxide/polybutylene terephtalate (PEO/PBTP); various copolymers such as polyhydroxy butyrate/valerate, polyethylene oxide/polybutylene terephtalate (PEO/PBTP), methyl methacrylate/2-hydroxy ethyl methacrylate, ethylene-vinyl acetate, laurylmethacrylate/methacryloylphosphoryl- choline, poly-L-glycolic acid (PLGA) and polyurethanes (PU); and biological polymers such as phosphorylcholine (PC), hyaluronic acide (HA), and fibrin.
  • biostable polymers such as polyethylene terephtalate (PET), polyure
  • the end-groups of the polymers can be blocked or unblocked. Suitable blocking groups include alkyl groups. Acceptable molecular weights for the biocompatible polymers can be determined by taking into consideration factors such as the desired polymer degradation rate, physical properties such as mechanical strength, and rate of dissolution of polymer in solvent. Typically, an acceptable range of molecular weight is between about 2,000 and 2,000,000 Daltons (Da). In a preferred embodiment, the polymer is a biodegradable polymer or copolymer.
  • the polymer is a poly(lactide-co-glycolide) (PLGA) with a lactide:glycolide ratio of about 1 :1 and a molecular weight between about 5,000 Da and 70,000 Da. In a more preferred embodiment, the molecular weight of the PLGA is between about 5,000 Da and 45,000 Da. As described in greater detail, a most preferred polymer is polyethyleneimine.
  • NOS nitric oxide synthase
  • the present invention also includes the use and/or incorporation of other agents in the polymeric matrix, in addition to NOS. Nearly any additional agent can be incorporated into the polymeric matrix, such as for example therapeutic, prophylactic, and diagnostic agents.
  • suitable therapeutic and/or prophylactic agents include proteins, such as hormones, antigens, and growth factors; nucleic acids, such as antisense molecules; and small molecules, such as antibiotics, steroids, decongestants, neuroactive agents, anesthetics and sedatives.
  • suitable diagnostic agents include radioactive isotopes and radiopaque agents.
  • the polymeric matrices can include more than one incorporated agent in addition to NOS.
  • thrombomodulin and heparans examples include thrombomodulin and heparans.
  • Thrombomodulin and heparans contribute to the non-thrombogenic properties of the endothelial cells.
  • Thrombomodulin which acts as a cofactor in the thrombin-driven activation of protein C, is a serine protease enzyme that plays a key role in the anticoagulation cascade.
  • a recombinant thrombomodulin can be combined with the various NOS- based coatings described herein.
  • the combination can be done either through direct cross-linking of the recombinant thrombomodulin terminal to an appropriate polymeric matrix in which the NOS enzyme resides, or introduced on the surface of specific liposome carriers, which can be cross-linked to a primary NOS-based coating.
  • a therapeutically, prophylactically, or diagnostically effective amount of the agents are incorporated into the polymeric matrices.
  • An effective amount of these agents can be readily determined by taking into consideration factors such as a body weight; age; physical condition; therapeutic, prophylactic, or diagnostic goal desired; type of agent used; type of polymer used; initial burst and subsequent release levels desired; and desired release rate.
  • the polymeric matrices will include between about 0.01 % (w/w) and 80% (w/w) of incorporated agent.
  • the incorporated agent in addition to NOS may be in the form of particles, for example, crystalline particles, non-crystalline particles, freeze dried particles, and lyophilized particles. Particles preferably are less than about 20 ⁇ m in size, and more preferably less than about 5 ⁇ m.
  • the particles also may include a stabilizing agent and/or other excipient.
  • the present invention coatings can be formed in nearly any fashion such that the NOS is incorporated within the polymeric matrix.
  • NOS is incorporated into the polymeric matrix by a layer-by-layer strategy in which a layer of either a polymeric material is formed or otherwise deposited on a substrate, such as the exterior surface of the medical device to be coated.
  • a layer of NOS or a layer of NOS and one or more other materials is deposited or otherwise applied onto the previously deposited polymeric material.
  • another layer of a polymeric material is deposited on or over, the previously deposited NOS- containing layer.
  • NOS-containing material or NOS itself
  • This process is repeated, preferably so that the number "n" of NOS- containing layers ranges from 1 to 50 or more, preferably from 2 to 20, and more preferably from 3 to 15.
  • the present invention includes layered assemblies in which adjacent layers of NOS or NOS-containing materials are separated by two or more layers of polymers, which as previously noted may have the same or different compositions.
  • a preferred method for forming the layered assemblies is to dip coat the substrate (and after an optional initial coating is deposited thereon, to thereby form an intermediate coated substrate) in either a solution of the desired polymer or a solution or mixture comprising NOS.
  • concentration of the NOS in the solution or mixture is generally from about 5 ⁇ M to about 100 ⁇ M, and preferably from about 20 to about 70 micromoles per milliliter.
  • the solution or mixture comprising NOS can include nearly any compatible liquid vehicle.
  • the time period for each dip coating operation may also vary, however generally a time period of from about 30 seconds to about 30 minutes is sufficient, with 10 minutes being preferred.
  • the polymer- containing solutions can be formed by known techniques.
  • a coating comprising NOS is formed upon a substrate as follows.
  • the substrate bearing an appropriate charge is dipped in polyethyleneimine polymer solution (typical concentration is 1.5 mg/ ml but this can vary) or in other suitably charged and biocompatible polymer.
  • polyethyleneimine polymer solution typically concentration is 1.5 mg/ ml but this can vary
  • the electrostatic interaction of the substrate surface with the polymer leaves a nanometer-thick layer bearing an overall charge of the polymer. Excess polymer (loosely/physically bound) is washed away with a wash liquid, such as deionized water.
  • the thickness of the polymeric layer on the substrate varies with the nature of the polymer used and concentration of the dipping solution.
  • thicknesses are on the order of 2 nm. Other thicknesses may be achieved, such as from about 0.1 nm to about 50 nm, and preferably from about 1 nm to about 10 nm.
  • the NOS is applied by forming a solution or mixture (or otherwise dispersing the NOS in one or more suitable liquid vehicles), and then depositing the solution/liquid onto the previously applied layer of polymer.
  • Preferred concentrations of NOS in this liquid vehicle range from 20 to 70 micromoles per ml.
  • the bulk concentration of NOS is not as important as the time of exposure to the surface. After exposure for about 10 min with the noted concentrations, the surface concentration of NOS molecules reaches a maximum, i.e. saturation.
  • the NOS solution can also contain other biomolecules that assist in counteracting thrombosis. If fact, in one embodiment, NOS is utilized in combination with thrombomodulin, an integral membrane protein that serves in the activation of protein C-dependent anticoagulation pathway. The combination of the nitric oxide release by NOS and thrombomodulin-derived anticoagulant effect strengthens the efficiency of the biomimetic coating.
  • the liquid vehicle is removed, optionally by drying, thereby leaving NOS.
  • Preferred thickness ranges for this layer are from 4 nm to 10 nm.
  • the polymer-coated substrate is simply dipped into the NOS-containing liquid and after a period of interaction, e.g. from about 5 to about 20 minutes, the substrate is removed, thoroughly washed, and dried before the next polymer deposition step.
  • NOS-containing layers a preferred range of NOS-containing layers is 5 to 10. Increasing the number of layers to 10 generally increases the efficiency of counteracting platelet aggregation as indicated by the LDH assay. This is detailed in the results of testing described herein.
  • the various layers are preferably electrostatically bound to each other, i.e. oppositely charged layers being attracted to each other. Other means of the attachment of the layers are conceivable and included in the present invention.
  • NOS nitric oxide
  • the resulting multi-layer coating which includes NOS within its interior, releases nitric oxide (NO) upon incorporation in a biological system, e.g. a human.
  • NOS is an enzyme, i.e., a biological catalyst, that uses the ingredients for NO synthesis from blood. As long as the supply of these ingredients continues and is accessible and proximate the NOS-containing coating, NO will be released from the coating. There is no depletion of NOS in the coating.
  • a final or outermost polymer layer as the "closing cap", since this practice prevents leaching of NOS from the top layer(s) of the multi-layer coating. It is conceivable that the final layer of the polymer may be crosslinked at the end of the layer-by-layer process to provide robustness of the film as a whole, which may also prevent potential leaching of the enzyme.
  • a preferred strategy for incorporating NOS into a polymeric matrix and forming a coating on a medical device is by the previously described layer-by-layer (LBL) construction.
  • LBL layer-by-layer
  • the present invention includes other strategies for incorporating NOS into a polymeric material and forming a coating on a medical device. It is also contemplated that NOS could be mixed or otherwise dispersed in the polymeric material(s) or such material(s) and one or more solvents, and then directly applied to form a coating.
  • NOS is incorporated in a layered array.
  • NOS is preferably dispersed in the array in layers, and most preferably, in sequentially deposited layers.
  • each NOS- containing layer is separated by one or more layer(s) of polymer.
  • Each NOS- containing layer is very thin, preferably on the order of only a few nanometers.
  • the relatively large NOS molecules are dispersed in a uniform fashion, or relatively so, over the area of the layer.
  • each NOS-containing layer is disposed between two polymer-containing layers. Most preferably, the NOS molecules are electrostatically bound to both the immediately adjacent polymer-containing layers.
  • the present invention provides a wide range of new opportunities and applications such as follows. It is to be understood that the present description of applications is merely representative, and in no way is the invention limited to these particular applications. [0082] Since nitric oxide has been shown to inhibit platelet aggregation, the nitric oxide-releasing polymeric materials of the invention are useful in laboratory and medical applications and procedures that involve contact with blood.
  • the NO- releasing polymeric material can be used in vivo, for example, to line or form blood- contacting surfaces of an in-body device such as a pacemaker, an implantable pulse generator (IPG), an implantable cardiac defibrillator (ICD), a pacemaker cardioverter defibrillator (PCD), a defibrillator, a spinal stimulator, a brain stimulator, a sacral nerve stimulator, a stent, a catheter, a lead, or a chemical sensor.
  • Examples of chemical sensors include optical or electrochemical sensors that can continuously monitor or measure physiologically important ions (H+, K+, Na+....etc.) and gases, such as CO 2 and O 2 , in the blood.
  • Ex vivo applications include incorporation of the nitric oxide releasing polymeric material into the blood-contacting surfaces of extracorporeal sensors and circulation devices such as blood oxygenators.
  • Another preferred application relates to extracorporeal membrane oxygenation (ECMO), which is a means in which blood is oxygenated outside the body.
  • ECMO takes over the work of the lungs and is often used for newborn babies whose lungs are failing despite other treatments. The procedure involves inserting plastic tubes or cannulae into the vein and artery of the neck and/or groin. The anticoagulant heparin is given to patients on ECMO to prevent clotting in the ECMO tubing and/or the development of clots on the membrane which could break off and migrate to the lungs or brain.
  • the nitric oxide-releasing polymer of the present invention has utility in combination with or as a substitute for heparin coatings and/or infusions to reduce or inhibit platelet aggregation or adherence. Similar problems with clotting of membranes and filters used in dialysis procedures can be solved by utilizing the preferred embodiment materials and coatings of the present invention.
  • Nitric oxide-releasing preferred embodiment polymers comprising NOS are useful for the treatment of many biological disorders.
  • the present invention provides methods of using such polymers or coatings.
  • a method of treating a mammal, e.g., a human, with a biological disorder treatable with nitric oxide is provided.
  • the method comprises administering to the mammal (e.g., human), in need thereof a preferred embodiment polymer comprising NOS, a composition thereof, or a polymer-containing medical device in an amount sufficient to treat the biological disorder in the mammal (e.g., human).
  • the method for treating a biological disorder in a mammal in which dosage with nitric oxide is beneficial comprises administering to a specific location on or within the mammal a medical device comprising NOS sufficient to cause release of nitric oxide.
  • the treatment can be prophylactic or therapeutic.
  • prophylactic is meant any degree in inhibition of the onset of the biological disorder, including complete inhibition.
  • therapeutic is meant any degree in inhibition of the progression of the biological disorder in the mammal (e.g., human).
  • biological disorder can be any biological disorder, so long as the disorder is treatable with nitric oxide. Suitable biological disorders include hypertension, restenosis, cancer, impotency, platelet aggregation, and a biological disorder due to a genetic defect or infection with an infectious agent, such as a virus, bacterium, fungus or parasite.
  • materials of the present invention can be used to promote the growth of new blood vessels and capillaries in a process known as angiogenesis.
  • the NO-releasing materials of the present invention may also be used to reduce inflammation and promote healing when used as a coating or substrate for implantable medical devices.
  • the present invention provides a method for promoting angiogenesis in a tissue of a mammal in need thereof.
  • the method comprises either applying or administering to the mammal a medical device comprising a nitric oxide-releasing polymer or coating comprising NOS to a specific location on or within the mammal in an amount effective to promote angiogenesis in the tissue.
  • Conditions that can be treated in accordance with this method of the present invention are characterized by insufficient vascularization (or predisposition thereto) of the affected tissue, i.e., conditions in which neovascularization is needed to achieve sufficient vascularization in the affected tissue, and include, for example, diabetic ulcers, gangrene, surgical or other wounds requiring neovascularization to facilitate healing; Buerger's syndrome; hypertension; ischemic diseases including, for example, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy, myocardial ischemia, ischemia of tissues such as, for example, muscle, brain, kidney and lung; and other conditions characterized by a reduction in microvasculature
  • Exemplary tissues in which angiogenesis can be promoted include: hypertension; ulcers (e.g., diabetic ulcers); surgical wounds; ischemic tissue, i.e., a tissue having a deficiency in blood as a result of an ischemic disease including, for example, muscle, brain, kidney and lung ischemia; ischemic diseases including, for example, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy, and myocardial ischemia.
  • ischemic tissue i.e., a tissue having a deficiency in blood as a result of an ischemic disease including, for example, muscle, brain, kidney and lung ischemia
  • ischemic diseases including, for example, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy, and myocardial ischemia.
  • nosocomial infections typically result from treatment in a hospital or healthcare service unit, and generally are secondary to a patient's original condition.
  • the preferred embodiment films and coatings are provided on a medical device or other object to be implanted into a patient's body.
  • the resulting device or object having the noted coating thereon exhibits an anti-microorganism effect which reduces the potential for infection resulting from implantation of the device.
  • PEI/NOS coatings or thin films described herein exhibit anti-microorganism effects upon a wide array of bacteria such as for example Pseudomonas aeruginosa.
  • Various other microorganisms are included such as viruses, fungi, microbes, parasites and other pathogens.
  • NOS-modified surfaces i.e. coatings, as described herein, control and ideally deter bacterial adhesion, growth, and/or existence upon the coated surfaces.
  • the NOS-based films and coatings described herein can be used to control bacterial adhesion, growth, and existence on short term and long term implantable devices.
  • Biofilms develop preferentially on inert surfaces, or on dead tissue, and occur commonly on medical devices and fragments of dead tissue such as dead bone; they can also form on living tissues, as in the case of endocarditis. Biofilms grow slowly, in one or more locations. And, biofilm infections are often slow to produce overt symptoms.
  • biofilm infections are rarely resolved by the host defense mechanisms. Antibiotic therapy typically reverses the symptoms caused by cells released from the biofilm, but fails to kill the biofilm. For this reason, biofilm infections typically exhibit recurring symptoms after cycles of antibiotic therapy, until the sessile population is surgically removed from the body. It is therefore preferable to prevent biofilm formation rather than to try to eradicate biofilms once they have formed.
  • the various coatings, thin films, and methods of the present invention are useful in the treatment of bacterial infection associated with biofilms, or in reducing the risk of a disease associated with biofilms, particularly biofilms caused by bacteria.
  • the various thin films may be used to coat devices that are inserted into an individual, e.g., a surgical device, catheter, prosthetic or other implant, to reduce the potential that the implanted device will develop a biofilm.
  • the coatings and thin films may be implanted to provide a high, localized concentration of the composition in the treatment of a localized infection.
  • the present invention composition may be provided in a deposit and formulated for sustained release.
  • Exemplary bacteria that can be targeted with the coatings and thin films of the present invention include, but are not limited to, Staphylococcus aureaus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus Group D, Clostridium perfringens, Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae.
  • the coatings and thin films of the invention effectively mediate the presence of bacteria such as Pseudomona and Staphylococcus.
  • aureus one of the leading causes of hospital acquired infections, causes a wide variety of diseases characterized by the formation of pus, including superficial and deep abscesses, empydema, meningitis, purulent arthritis, and septicemia and endocarditis. In addition, this species causes two toxinoses: food poisoning and exfolative skin disease.
  • the coatings and thin films of the present invention exhibit an ability to reduce the number of bacterial colonies by at least 10%. More preferably, the coatings of the invention can reduce the number of bacterial colonies by at least 25% and even more preferably by 50%, 75%, 85%, 95%, and up to 100%. All individual values and ranges falling between these ranges and values are within the scope of the present invention. [0095]
  • the present invention also provides a means for treating a variety of fungal infections associated with an implantable medical device.
  • fungal infections include, but are not limited to, asthma, chronic rhinosinusitis, allergic fungal sinusitis, sinus mycetoma, non-invasive fungus induced mucositis, non-invasive fungus induced intestinal mucositis, chronic otitis media, chronic colitis, inflammatory bowel diseases, ulcerative colitis, Crohn's disease, candidemia, intraabdominal abscesses, peritonitis, pleural space infections, esophageal candidiasis and invasive aspergillosis.
  • Exemplary fungi that can be treated using the preferred embodiment coatings of the invention include, without limitation, Absidia, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus terreus, Aspergillus versicolor, Altemaria, Basidiobolus, Bipolaris, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lypolytica, Candida parapsilosis, Candida tropicalis, Cladosporium, Conidiobolus, Cunninahamella, Curvularia, Dreschlera, Exserohilum, Fusarium, Malbranchia, Paecilonvces, Penicillium, Pseudallascheria, Rhizopus, Schizophylum, Sporothrix, Acremonium, Arachniotus citrinus, Aurobasidioum,
  • the present invention can be applied and/or utilized in nearly any biological system in which NOS facilitates the release and/or generation of nitric oxide.
  • biological systems will be mammals, and preferably humans.
  • human application is a primary objective of the present invention, it is contemplated that the invention can be used in a wide assortment of animals such as dogs, horses, livestock, etc.
  • a pyrolytic graphite (PG) surface (unless indicated otherwise) was initially modified utilizing a diazonium grafting method as described in Blankespoor, R.; Limoges, B.; Schollhorn, B.; Syssa-Magale, J. L.; Yazidi, D., Dense monolayers of metal-chelating ligands covalently attached to carbon electrodes electrochemically and their useful application in affinity binding of histidine-tagged proteins. Langmuir 2005, 21 , (8), 3362-75. With the right diazonium derivative, this process yields a uniform negative charge at the surface.
  • the PG surface is dipped in a mixture of 50 mM p-aminobenzoate and 50 mM sodium nitrite at 4°C, in a 3 necked electrochemical cell.
  • the potential is scanned between 0 and -0.6 V vs Ag/AgCI leading to the covalent attachment of diazonium group on the surface, with a free carboxylic group (COO " ).
  • the pyrolytic graphite surface was alternatively dipped coated in a polyethyleneimine (PEI; 1.5 mg/ml) solution and iNOS solution for 10 minutes respectively to achieve the desired film composition.
  • PEI polyethyleneimine
  • iNOS solution iNOS
  • ITO indium-doped tin oxide
  • the ITO slides were initially modified utilizing a diazonium method as described in the previously noted article by Blankespoor et al. to obtain a uniform negative charge at the surface.
  • An LBL modification was performed at the surface of the ITO slide as previously described herein under the heading "Preparation of PEI/NOS Film.”
  • the enzyme iNOS has a characteristic soret band at 421 nm, typical of this specific class of heme proteins. The soret band absorbance was monitored as a function of layers of enzyme deposited. Five layers of NOS enzyme were deposited in between alternate layers of PEI polymer.
  • Figure 1 indicates the absorbance spectra at 421 nm with each layer of enzyme deposited at the surface of modified indium tin oxide (ITO) glass slides.
  • the increased absorbance seen with each cycle of NOS deposition within the film indicates the LBL method of preparing those films allows for increased enzyme loading.
  • the control of film composition can be achieved with the LBL method, which is critical to optimizing NO release from those films.
  • the infrared spectra of polypeptides exhibit a number of specific features called amide bands that represent different vibrational modes of the peptide bond.
  • the amide I band at around 1650 cm "1 , is most widely used for secondary structure analyses.
  • Figure 2 reveals the IR spectrum of NOS enzyme embedded within two PEI layers at the surface of the ATR crystal. A significant band at 1650 cm "1 typical of the amide I band is seen.
  • the amide I band is an indication of the retention of the secondary structures of proteins. In this case, the structural integrity of the NOS enzyme within the film environment is demonstrated, a prelude to retention of catalytic function within the film.
  • Electrochemical characterization was carried out at the surface of pyrolytic graphite (PG) electrodes. Prior to coating, basal-plane PG electrodes were polished consecutively on 400 grit carbimet disks (Buehler), then on Buehler microcloth, using 0.3 ⁇ m alumina. Electrodes were then ultrasonicated in pure water for 30s, rinsed, and dried in air. PG electrodes were oxidized as described in previously. Typically, PG electrodes are modified using the diazonium method as previously described. The surface of the electrodes is modified as described above. All experiments were performed at room temperature in 100 mM pH 7.0 phosphate buffer, unless otherwise indicated.
  • the buffer was purged with purified nitrogen for at least 30 min prior to the experiments to remove dioxygen. A nitrogen blanket was then kept over the solution throughout the experiments. A fresh solution of 2 mM NO was prepared, and aliquots were injected into the system. NO stock-solutions were made by bubbling pure NO gas through degassed water as described in Zhang, X. B., M, Amperometric detection of Nitric Oxide. Mod. Asp. Immunobiol. 2000, 1 , (4), 160-165. Typically deionized water in a small vial capped with a rubber septum is degassed with purified nitrogen. The nitrogen was previously passed through an alkaline pyrogallol (5% w/v) solution to scavenge any traces of oxygen.
  • NO gas was bubbled through for 30 min.
  • the NO gas used was purified by passing the gas through the 5%-pyrogallol solution in saturated potassium hydroxide to remove trace oxygen and then through a 10% (w/v) potassium hydroxide solution to remove potential other nitrogen oxides (NO x ).
  • the concentration of the NO in the saturated water was 2 mM which was independently confirmed via a photometric method based on the stoichiometric conversion of oxyhemoglobin to methemoglobin by NO.
  • the stock and NO standard solutions can be freshly prepared through serial dilution of the NO saturated solution prior to each experiment.
  • Cyclic Voltammetry of PEI/NOS film at the surface of PG electrodes illustrates an irreversible catalytic wave at -0.9 V/s, a response typical to the catalytic reduction of NO by Myoglobin, and other P450 enzymes.
  • An increase in catalytic currents is observed with increasing NO concentrations.
  • a negative control experiment (PEI film devoid of NOS), does not show a catalytic current at -0.9 V/s at the same NO concentrations (results not shown).
  • the electrochemical reduction of NO at the surface of the PEI/NOS film is indicative of the retention of iNOS catalytic activity within the polymeric environment. Quartz Crystal Microbalance
  • Atomic Force Microscopy characterization was carried out at the surface of 1cm x 1cm highly oriented pyrolytic graphite (HOPG) slides. Initially, the top layer on the slide was peeled to eliminate any interference from dust particles. The HOPG surface was modified with the diazonium method as previously described. The surface was modified as previously described. Figures 5-7 illustrate the 3D AFM images of the layer-by-layer deposition of the polyethyleneimine (PEI) polymer and the enzyme Nitric Oxide Synthase at the surface of the HOPG slide (the 3D AFM image of bare HOPG support showing the typical, and almost atomically- smooth, initial surface prior to coating is not shown here).
  • PEI polyethyleneimine
  • Figure 5 illustrates the 3D AFM image of the deposition of a layer of the polymer PEI at the surface of the HOPG slide.
  • the average surface height is of the order of 2.0 nm.
  • Figure 6 illustrates the 3D AFM image of the deposition of a layer of NOS over a layer of the polymer PEI at the surface of the HOPG slide. Consistent with QCM data, the figure shows a uniform distribution of the enzyme over the surface. The average surface height is of the order of 8.0 nm, consistent with known NOS dimensions.
  • Figure 7 shows the 3D AFM image of the same HOPG slide after the sequential deposition of PEI over a layer of NOS resulting in the PEI/NOS/PEI coating.
  • the biocompatible polymeric coating now brings the average surface height to the order of 10.0 nm.
  • the AFM images provide evidence of a uniform coverage of the modified surface in question using the LBL deposition of PEI and NOS enzyme, which is critical to its potential application as a surface coating of implantable devices coming in contact with blood.
  • the films were prepared as described above.
  • NADPH 150 ⁇ M was used as a source of reducing equivalents, which is widely used in the NOS community.
  • the enzymatic reaction was run at 37°C for the desired time period.
  • the enzymatic activity of the film at the pyrolytic graphite surface or the surface of other materials was quantified by measuring NO flux using the Griess assay.
  • Typical substrates prepared as described above are utilized to investigate the NO production capability from the trapped NOS enzymes.
  • the enzymatic activity of the coatings at the pyrolytic graphite surface was quantified by measuring NO flux using the Griess assay.
  • Figure 9 reveals surface NO fluxes from films composed of 5 layers of PEI/NOS.
  • Figure 10 shows increased NO fluxes from a film composed of 5 layers of PEI/NOS relative to a film preparation of 3 layers, and 1 layer of PEI/NOS respectively, indicating that higher NO fluxes can be achieved by increasing the thickness, i.e. enzyme loading, of the polymeric coatings.
  • the example of NOS based polyethyleneimine coatings developed with the layer-by- layer methodology provide evidence that NOS-based biocompatible coatings as described herein are capable of releasing NO at levels that can be brought (adjusted) to mimic stimulated human endothelial cells.
  • the NO release from the PEI films example exhibited a two-phase kinetic: an initial burst and then a slower sustained release over a period of 4 days.
  • the rate of NO release is crucial to the optimum effect of NO released from the PEI films on the surrounding tissues.
  • the optimum rate of NO can be adjusted for the particular biocompatible matrix holding NOS enzymes and the method used to deposit the coating.
  • NO release from the PEI films was deemed to be tunable with respect to the number of layers of iNOS enzyme embedded in between alternate PEI layers.
  • 150 ⁇ l_ of lysing buffer was incubated in each well for 1 h at 37 ° C with occasional agitation to completely disrupt the platelet membranes. Then, 100 ⁇ l_ of each lysate solution was pipetted into wells of a 96-well polystyrene microtiter plate (Fisher) that contained 100 ⁇ l_ of reagent from an LDH assay kit (Roche Applied Sciences, Indianapolis, IN), and incubated for 30 min at 25°C. Then, 50 ⁇ l_ of stop solution was added and absorbance of each well at 490 nm was monitored by a Labsystems Multiskan RC microplate reader (Fisher).
  • LDH assay is useful in quantifying cell and platelet adhesion at the surface of the PEI/NOS coatings.
  • LDH is an enzyme found in cells at levels proportional to its size, and is released following cell breakdown. In vitro determination of the degree of platelet adhesion to surfaces using LDH assay has been reported.

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Abstract

L'invention porte sur un revêtement biocompatible. Le revêtement comprend l'enzyme oxyde nitrique synthase, qui libère de l'oxyde nitrique ou conduit à la libération d'oxyde nitrique, et une matrice biocompatible incorporant l'enzyme oxyde nitrique synthase. Le revêtement peut être appliqué sur des dispositifs médicaux. Divers exemples de compositions, de matériaux, de procédés et de dispositifs utilisant ou autrement associés à de tels revêtements sont également divulgués.
PCT/US2008/085224 2007-12-03 2008-12-02 Revêtements à libération d'oxyde nitrique incorporant une enzyme oxyde nitrique synthase Ceased WO2009073643A2 (fr)

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US8282967B2 (en) 2005-05-27 2012-10-09 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
CN102961783A (zh) * 2012-04-20 2013-03-13 南开大学 抗凝血性人工血管支架材料的构建方法
US8591876B2 (en) 2010-12-15 2013-11-26 Novan, Inc. Methods of decreasing sebum production in the skin
US8981139B2 (en) 2011-02-28 2015-03-17 The University Of North Carolina At Chapel Hill Tertiary S-nitrosothiol-modified nitric—oxide-releasing xerogels and methods of using the same
US9526738B2 (en) 2009-08-21 2016-12-27 Novan, Inc. Topical gels and methods of using the same
US9919072B2 (en) 2009-08-21 2018-03-20 Novan, Inc. Wound dressings, methods of using the same and methods of forming the same
EP4223512A4 (fr) * 2020-09-30 2025-02-26 Daicel Corporation Stratifié

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US6379691B1 (en) * 1998-09-29 2002-04-30 Medtronic/Ave, Inc. Uses for medical devices having a lubricious, nitric oxide-releasing coating
US6270779B1 (en) * 2000-05-10 2001-08-07 United States Of America Nitric oxide-releasing metallic medical devices
US20030228564A1 (en) * 2001-05-30 2003-12-11 Edrich Richard Alan Nitric oxide in a pathogen inactivation process
US20040248323A1 (en) * 2003-06-09 2004-12-09 Protometrix, Inc. Methods for conducting assays for enzyme activity on protein microarrays
US20060039950A1 (en) * 2004-08-23 2006-02-23 Zhengrong Zhou Multi-functional biocompatible coatings for intravascular devices
US8734851B2 (en) * 2005-04-29 2014-05-27 Wisconsin Alumni Research Foundation Localized delivery of nucleic acid by polyelectrolyte assemblies

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US9403851B2 (en) 2005-05-27 2016-08-02 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
US11691995B2 (en) 2005-05-27 2023-07-04 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
US8282967B2 (en) 2005-05-27 2012-10-09 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
US8956658B2 (en) 2005-05-27 2015-02-17 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
US8962029B2 (en) 2005-05-27 2015-02-24 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
US9403852B2 (en) 2005-05-27 2016-08-02 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
US9526738B2 (en) 2009-08-21 2016-12-27 Novan, Inc. Topical gels and methods of using the same
US9737561B2 (en) 2009-08-21 2017-08-22 Novan, Inc. Topical gels and methods of using the same
US9919072B2 (en) 2009-08-21 2018-03-20 Novan, Inc. Wound dressings, methods of using the same and methods of forming the same
US10376538B2 (en) 2009-08-21 2019-08-13 Novan, Inc. Topical gels and methods of using the same
US11583608B2 (en) 2009-08-21 2023-02-21 Novan, Inc. Wound dressings, methods of using the same and methods of forming the same
US8591876B2 (en) 2010-12-15 2013-11-26 Novan, Inc. Methods of decreasing sebum production in the skin
US8981139B2 (en) 2011-02-28 2015-03-17 The University Of North Carolina At Chapel Hill Tertiary S-nitrosothiol-modified nitric—oxide-releasing xerogels and methods of using the same
US9713652B2 (en) 2011-02-28 2017-07-25 The University Of North Carolina At Chapel Hill Nitric oxide-releasing S-nitrosothiol-modified silica particles and methods of making the same
CN102961783A (zh) * 2012-04-20 2013-03-13 南开大学 抗凝血性人工血管支架材料的构建方法
EP4223512A4 (fr) * 2020-09-30 2025-02-26 Daicel Corporation Stratifié

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